Reverse beta oxidation pathway

ABSTRACT

The invention relates to recombinant microorganisms that have been engineered to produce various chemicals using genes that have been repurposed to create a reverse beta oxidation pathway. Generally speaking, the beta oxidation cycle is expressed and driven in reverse by modifying various regulation points for as many cycles as needed, and then the CoA thioester intermediates are converted to useful products by the action of termination enzymes.

PRIOR RELATED APPLICATIONS

This application is a National Phase filing under 35 U.S.C. §371 ofInternational Application PCT/US2012/024051 filed on Feb. 7, 2012 whichclaims priority to 61/440,192, filed Feb. 7, 2011, Both applications areexpressly incorporated herein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

This invention was made with government support under CBET-1134541 andCBET-1067565 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to recombinant microorganisms that have beenengineered to produce alcohols carboxylic acids, alkanes or alkenes,using genes that have been repurposed to create a reverse beta oxidationpathway.

Generally speaking, the beta oxidation cycle is driven in reverse for asmany cycles as needed, and then the CoA thioester intermediates can thenbe converted to useful products by the action of different types oftermination enzymes: i) thioesterases, or acyl-CoA:acetyl-CoAtransferases, or phosphotransacylases and carboxylate kinases (whichform carboxylic acids) or ii) alcohol-forming coenzyme-A thioesterreductases (which make alcohols) or iii) aldehyde-forming CoA thioesterreductases and alcohol dehydrogenases (which together form alcohols) oriv) aldehyde-forming CoA thioester reductases and aldehydedecarbonylases (which together form alkanes or terminal alkenes) or v)olefin-forming enzymes (such as OleA, OleB, OleC, OleD, which togetherform internal alkenes or terminal alkenes or trienes or alkenols).

The carboxylic acids include monocarboxylic acids, β-keto acids,β-hydroxy acids, and trans-Δ²-fatty acids of different chain lengths.The alcohols include n-alcohols, β-keto alcohols, 1,3-diols, andtrans-Δ²-alcohols of different chain lengths. Alkanes include aliphaticalkanes of different chain lengths. Aliphatic alkenes (also calledolefins) include terminal olefins, internal olefins, trienes, andalkenols.

BACKGROUND OF THE INVENTION

In recent years much effort has been devoted to the biologicalproduction of renewable fuels such as ethanol. However, ethanol is notan ideal fuel, suffering from problems such as high hygroscopicity, highvapor pressure and low energy density. These qualities make ethanolincompatible with the current facilities used in the storage,distribution and use of liquid transportation fuels.

Higher-chain (C≧4) alcohols (e.g. n-butanol), fatty acid methyl esters(FAMEs) and hydrocarbons (alkanes and alkenes) offer several advantagescompared to ethanol, including reduced hygroscopicity, reducedvolatility, and higher energy density. These qualities make n-butanoland other higher alcohols more compatible with our currentinfrastructure for storage, distribution and usage.

The aforementioned long-chain fuels and chemicals are generated fromshort-chain metabolic intermediates through pathways that requirecarbon-chain elongation. However, biological efforts to date have beenless than satisfactory, particularly where non-native genes areintroduced to drive synthesic of longer chain molecules.

Therefore, what is needed in the art are better biological methods ofmaking higher-chain (C≧4) fuels (e.g. alcohols, fatty acid methylesters, FAMEs, and hydrocarbons) that are more efficient and costeffective than are currently available. The ideal method would alsoallow the production of chemicals, such as carboxylic acids andalcohols, that can be used as feedstocks in other industries.

SUMMARY OF THE INVENTION

We have developed an alternative approach to engineering microbes tomake chemicals, such as alcohols, carboxylic acids, alkanes, andalkenes, that uses a functional reversal of the β-oxidation cycle as ametabolic platform for the synthesis of alcohols and carboxylic acidswith various chain lengths and functionalities (FIG. 1A).

This pathway operates with coenzyme-A (CoA) thioester intermediates anddirectly uses acetyl-CoA for acyl-chain elongation (rather than firstrequiring ATP-dependent activation to malonyl-CoA), characteristics thatenable product synthesis at maximum carbon and energy efficiency. Thereversal of the β-oxidation cycle was engineered in Escherichia coli andused in combination with endogenous dehydrogenases and thioesterases tosynthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- andtrans-Δ2-carboxylic acids.

The superior nature of the engineered pathway was demonstrated byproducing higher-chain linear n-alcohols (C≧4) and extracellularlong-chain fatty acids (C>10) at higher efficiency than previouslyreported. The ubiquitous nature of β-oxidation, aldehyde/alcoholdehydrogenase, thioesterase, decarbonylase enzymes has the potential toenable the efficient synthesis of these products in other industrialorganisms such as S. cerevisiae, Z. mobilis, B. subtilis, etc.

Although we have exemplified n-butanol, 4-C 3-hydroxy-, 3-keto- andtrans-Δ2-carboxylic acids, longer-chain (C>4) n-alcohols, and long-chain(C>10) fatty acids herein, we have also shown that with judicious use ofstarting materials and enzymes, we can also make other carboxylic acids,alcohols, alkanes and alkenes, depending on which termination enzymesare overexpressed in the engineered microorganism.

We have already engineered a one-turn reversal of the beta-oxidationcycle for the production of n-butanol in E. coli, an organism consideredunable to produce this alcohol in the absence of foreign genes. Thisapproach, called herein an “endogenous” or “native” gene approach, isnot based on transferring pathways from organisms that naturally producen-butanol, but native genes and proteins are repurposed to producebutanol by manipulation of pathways.

In general, our methodology to drive the reversed β-oxidation cycleinvolves the following three steps: 1) functionally expressing the betaoxidation cycle in the absence of its naturally inducing substrates(i.e. absence of fatty acids) and presence of a non-fatty acid carbonsource (e.g. presence of glucose); 2) driving the beta oxidation cyclein the reverse/biosynthetic direction (as opposed to its naturalcatabolic/degradative direction); and 3) expressing termination enzymesthat act on the appropriate intermediate of the beta oxidation cycle tomake desired products.

In more detail, the recombinant engineering is:

1) Express the β-Oxidation Cycle in the Absence of its NaturallyInducing Substrates (i.e. Absence of Fatty Acids) and Presence of aNon-Fatty Acid Carbon Source (e.g. Presence of Glucose):

In order to express the β-oxidation cycle, first i) mutations fadR andatoC(c) enable expression of the genes encoding beta oxidation enzymesin the absence of fatty acids; ii) an arcA knockout (ΔarcA) enables theexpression of genes encoding beta oxidation cycle enzymes/proteins underanaerobic/microaerobic conditions (microaerobic/anaerobic conditions areused in the production of fuels and chemicals but lead to repression ofbeta oxidation genes by ArcA); and iii) replacement of native cyclic AMPreceptor protein (crp) with a cAMP-independent mutant (crp*) enables theexpression of genes encoding beta oxidation cycle enzymes/proteins inthe presence of a catabolite-repressing carbon source such as glucose(glucose is the most widely used carbon source in fermentation processesand represses the beta oxidation genes).

2) Driving the Beta Oxidation Cycle in the Reverse/BiosyntheticDirection (as Opposed to its Natural Catabolic/Degradative Direction).

In addition to functionally expressing the β-oxidation cycle, we proposethe following modifications to achieve the reverse operation of thispathway: iv) the use of microaerobic/anaerobic conditionsprevents/minimizes the metabolism of acetyl-CoA through thetricarboxylic acids (TCA) cycle and makes acetyl-CoA available to drivethe beta oxidation cycle in the reverse/biosynthetic direction; v) pta(or ackA or both), poxB, adhE, yqhD, and eutE knockouts block/reduce thesynthesis of acetate (Δpta or ΔackA and poxB) and ethanol (ΔadhE, ΔyqhD,and ΔeutE) from acetyl-CoA and therefore make acetyl-CoA available todrive the beta oxidation cycle in the reverse/biosynthetic direction;vi) overexpression of thiolases, the first step in the reversal of thebeta oxidation cycle, enable the channeling of acetyl-CoA into thispathway and hence its operation in the reverse direction; vii) ldhA,mgsA, and frdA knockouts block/reduce the synthesis of lactate (ΔldhAand ΔmgsA) and succinate (ΔfrdA) from pyruvate and phosphoenolpyruvate,respectively, making more phosphoenolpyruvate and pyruvate available forthe synthesis acetyl-CoA and therefore making acetyl-CoA available todrive the beta oxidation cycle in the reverse/biosynthetic direction;viii) overexpression of pyruvate:flavodoxin oxidoreductase (ydbK) andacyl-CoA dehydrogenase (ydiO and ydiQRST) enables the coupling ofpyruvate oxidation (pyruvate→acetyl-CoA+CO₂+Fd_(red)) andtrans-Δ²-enoyl-CoA reduction (trans-Δ²-enoyl-CoA+Fd_(red)→acyl-CoA) andhence drive the beta oxidation in the reverse direction.

3) Conversion of CoA Thioester Intermediates to the Desired EndProducts.

Generally speaking, there are several termination enzymes that will pullreaction intermediates out the reverse β-oxidation cycle and produce thedesired end product (FIG. 1A):

i) CoA thioester hydrolases/thioesterases, or acyl-CoA:acetyl-CoAtransferases, or phosphotransacylases and carboxylate kinases forcarboxylic acids (i.e. short, medium, and long-chain monocarboxylicacids, β-keto acids, β-hydroxy acids, trans-Δ²-fatty acids),

ii) alcohol-forming CoA thioester reductases for alcohols (i.e. short,medium, and long-chain n-alcohols, β-keto alcohols, 1,3-diols,trans-Δ²-alcohols),

iii) aldehyde-forming CoA thioester reductases and alcoholdehydrogenases which together form alcohols (i.e. short, medium, andlong-chain n-alcohols, β-keto alcohols, 1,3-diols, trans-Δ²-alcohols).One or more of these termination enzymes can be overexpressed, as neededdepending on the desired end product.

iv) aldehyde-forming CoA thioester reductases and aldehydedecarbonylases (which together form alkanes or terminal alkenes ofdifferent chain lengths), and

v) olefin-forming enzymes (which together form aliphatic internalalkenes or terminal alkenes or trienes or alkenols).

The termination enyzmes can be native or non-native as desired forparticular products, but it is preferred that the reverse beta oxidationcycle use native genes.

4. Regulation of Product Chain Length.

The chain length of thioester intermediates determines the length of endproducts, and can be controlled by using appropriate termination enzymeswith the desired chain-length specificity. Additionally, chainelongation can be inhibited or promoted by reducing or increasing theactivity of thiolases with the desired chain-length specificity. Thesetwo methods can be used together or independently. For example:

i) knockout of fadA, fadI, and paaJ to avoid chain elongation beyond1-to-2 turns of the cycle (generates 4- & 6-carbon intermediates andproducts, or 5- & 7-carbon intermediates and products, depending on theuse of acetyl-CoA or propionyl-CoA as primer/starter molecule) andoverexpression of the short-chain thiolases yqeF or atoB or short chainsalcohol dehydrogenases such as fucO or yqhD;

ii) overexpression of fadB, fadI, and paaJ to promote chain elongationand overexpression of long-chain thiolases tesA, tesB, fadM, ybgC oryciA or long chain alcohol dehydrogenases such as ucpA, ybbO, yiaY,betA, ybdH or eutG; The term “appropriate” is used herein to refer to anenzyme with the required specificity toward a given intermediate (i.e.acyl-CoA, enoyl-CoA, hydroxyacyl-CoA, and ketoacyl-CoA) of a specificchain length. Please note that the chain length of the thioesterintermediates can be controlled by manipulating thiolases (as describedabove), and hence only thioesters of the desired chain length will beavailable to the termination enzymes.

Abbreviation Definition Δ Refers to reduced activity wherein reducedactivity is at least an 75% reduction of wild type activity, andpreferably, 80, 85, 90, 95 or 100% reduction. 100% reduction in activitymay also be called knockout or null mutant herein. ackA Gene encodingacetate kinase, required for synthesis of acetate from acetyl-CoA. acr1Gene encoding a fatty aldehyde-forming acyl-CoA reductases fromAcinetobacter calcoaceticus acrM Gene encoding a fatty aldehyde-formingacyl-CoA reductases from Acinetobacter sp. strain M-1 adhE Gene encodingaldehyde/alcohol dehydrogenase, required for synthesis of ethanol fromacetyl-CoA arcA Encodes the cytosolic transcription factor of E. coli'sArcAB two-component system, a global regulator of gene expression undermicroaerobic and anaerobic conditions. ArcAB regulates the expression ofa large number of operons involved in respiratory and fermentativemetabolism (including repression of fad regulon). arcB Encodes themembrane associated sensor kinase and phosphatase of E. coli's ArcABtwo-component system, a global regulator of gene expression undermicroaerobic and anaerobic conditions. ArcAB regulates the expression ofa large number of operons involved in respiratory and fermentativemetabolism (including repression of fad regulon). atoB Gene encoding anacetyl-CoA acetyltransferase atoC Encodes the cytosolic transcriptionfactor of E. coli's AtoSC two-component system, which induces the atooperon (atoDAEB) for metabolism of short chain fatty acids in responseto the presence of acetoacetate. atoC(c) atoC mutant that inducesconstitutive expression of the ato operon (atoDAEB) in the absence ofacetoacetate. atoS Encodes the membrane associated sensor kinase of E.coli's AtoSC two- component system, which induces the ato operon(atoDAEB) for metabolism of short chain fatty acids in response to thepresence of acetoacetate. betA Gene encoding choline dehydrogenase; usedas a surrogate of alcohol dehydrogenase in the synthesis of n-alcoholscrp Encodes transcriptional dual regulator CRP, which upon binding toits allosteric effector cyclic AMP (cAMP) regulate the expression ofabout 200 genes (most of them involved in the catabolism of carbonsources, including the fad regulon). crp* crp mutant encoding acAMP-independent CRP (i.e. CRP*, which does not require cAMP to regulategene expression and hence prevents catabolite repression of fad regulonin the presence of glucose) cysJ Gene encoding the flavoprotein subunitcomplex of sulfite reductase. Along with YdbK, CysJ could form apyruvate: NADP oxidoreductase: we propose that ydbK and cysJ wouldencode the N-terminal pyruvate: ferredoxin oxidoreductase domain and theC-terminal NADPH-cytochrome P450 reductase domain, respectively. EgterGene encoding an NAD(P)H-dependent transenoyl-CoA reductase from T.gracilis eutE Gene encoding predicted aldehyde dehydrogenase with highsequence similarity to adhE eutG Gene encoding predicted alcoholdehydrogenase fadA Gene encoding 3-ketoacyl-CoA thiolase (thiolase I),component of fatty acid oxidation complex fadB Gene encodinghydroxyacyl-CoA dehydrogenase, aka fused 3- hydroxybutyryl-CoA epimeraseand delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase and enoyl-CoAhydratase, part of fatty acid oxidation complex fadBA Both fadB and fadAfadD Gene encoding acyl-CoA synthetase (long-chain-fatty-acid--CoAligase), part of fatty acyl-CoA synthetase complex fadE Gene encodingacyl-CoA dehydrogenase, a medium-long-chain fatty acyl- CoAdehydrogenase fadI Gene encoding 3-ketoacyl-CoA thiolase, part of fattyacid oxidation complex fadJ Gene encoding hydroxyacyl-CoA dehydrogenase,aka fused enoyl-CoA hydratase and epimerase and isomerase fadK Geneencoding short chain acyl-CoA synthetase fadL Gene encoding long-chainfatty acid outer membrane transporter fadM Gene encoding long-chainacyl-CoA thioesterase fadR Gene encoding a dual regulator of fatty acidmetabolism, which exerts negative control over the fad regulon andpositive control over expression of unsaturated fatty acid biosynthesisgenes fadR fadR mutant that allows expression of the fad regulon in theabsence of fatty acids fnr Gene encoding transcriptional dual regulator,regulates genes involved in the transition from aerobic to anaerobicgrowth frdA Gene encoding fumarate reductase, required for synthesis ofsuccinate from fumarate fucO Gene encoding L-1,2-propanedioloxidoreductase ldhA Gene encoding lactate dehydrogenase mgsA Geneencoding methylglyoxal synthase; key enzyme in the synthesis of lactatethrough the methylglyoxal bypass oleA Gene encoding the enzyme thatcatalyzes non-decarboxylative Claisen condensation of CoA-thioesters inXanthomonas campestris oleB Gene encoding a member of the α/β-hydrolasesuperfamily in Xanthomonas campestris oleC Gene encoding a member of theAMPdependent ligase/synthase superfamily or acetyl-CoA synthetase-likesuperfamily in Xanthomonas campestris oleD Gene encoding a member of theshort-chain dehydrogenase/reductase superfamily in Xanthomonascampestris paaJ β-ketoadipyl-CoA thiolase catalyzing two beta-oxidationsteps in phenylacetate catabolism PCC7942_orf1593 Gene encoding analdehyde decarbonylase from Synechococcus elongatus PCC7942 pmAD Geneencoding an aldehyde decarbonylase from Prochlorococcus marinus MIT9313poxB Gene encoding pyruvate oxidase, which catalyzes the oxidativedecarboxylation of pyruvate to form acetate, reduced ubiquinone(ubiquinol), and CO₂ pta Gene encoding phosphotransacetylase, requiredfor synthesis of acetate from acetyl-CoA Tdter Gene encoding anNAD(P)H-dependent transenoyl-CoA reductase from T. denticola tesA Geneencoding multifunctional acyl-CoA thioesterase I and protease I andlysophospholipase L1 tesB Gene encoding thioesterase II ucpA predictedoxidoreductase, sulfate metabolism protein (used as surrogate ofaldehyde-forming acyl Coenzyme A Reductase) ybbO predictedoxidoreductase with NAD(P)-binding Rossmann-fold domain (used assurrogate of aldehyde-forming acyl Coenzyme A Reductase) yciA Geneencoding acyl-CoA thioesterase ydiL Gene encoding fused predictedacetyl-CoA: acetoacetyl-CoA transferase: α subunit/β subunit ydiO Genesencoding predicted acyl-CoA dehydrogenase ydiQ Gene encoding putativesubunit of YdiQ-YdiR flavoprotein ydiR Gene encoding putative subunit ofYdiQ-YdiR flavoprotein ydiS Gene encoding putative flavoprotein ydiTGene encoding putative ferredoxin yiaY Gene encoding predictedFe-containing alcohol dehydrogenase yqeF Gene encoding predictedacetyl-CoA acetyltransferases yqhD Gene encoding NADP-dependentaldehyde/alcohol dehydrogenase + Refers to an overexpressed activity,meaning at least 150% wild type activity, and preferably 200, 500, 1000%or more.

As used herein, references to cells or bacteria or strains and all suchsimilar designations include progeny thereof. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations that have been added to the parent.Mutant progeny that have the same function or biological activity asscreened for in the originally transformed cell are included. Wheredistinct designations are intended, it will be clear from the context.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally coupled nucleic acid sequences.

As used herein “recombinant” is relating to, derived from, or containinggenetically engineered material. In other words, the genome wasintentionally manipulated in some way.

“Reduced activity” or “inactivation” is defined herein to be at least a75% reduction in protein activity, as compared with an appropriatecontrol species. Preferably, at least 80, 85, 90, 95% reduction inactivity is attained, and in the most preferred embodiment, the activityis eliminated (100%, aka a “knock-out” or “null” mutants). Proteins canbe inactivated with inhibitors, by mutation, or by suppression ofexpression or translation, and the like. Use of a frame shift mutation,early stop codon, point mutations of critical residues, or deletions orinsertions, and the like, can completely inactivate (100%) gene productby completely preventing transcription and/or translation of activeprotein.

“Overexpression” or “overexpressed” is defined herein to be at least150% of protein activity as compared with an appropriate controlspecies, and preferably 200, 500, 1000% or more. Overexpression can beachieved by mutating the protein to produce a more active form or a formthat is resistant to inhibition, by removing inhibitors, or addingactivators, and the like. Overexpression can also be achieved byremoving repressors, adding multiple copies of the gene to the cell, orupregulating the endogenous gene, and the like.

The term “endogenous” or “native” means that a gene originated from thespecies in question, without regard to subspecies or strain, althoughthat gene may be naturally or intentionally mutated, or placed under thecontrol of a promoter that results in overexpression or controlledexpression of said gene. Thus, genes from Clostridia would not beendogenous to Escherichia, but a plasmid expressing a gene from E. colior would be considered to be endogenous to any genus of Escherichia,even though it may now be overexpressed.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims or the specification means one or more thanone, unless the context dictates otherwise.

The term “about” means the stated value plus or minus the margin oferror of measurement or plus or minus 10% if no method of measurement isindicated.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or if thealternatives are mutually exclusive.

The terms “comprise”, “have”, and “include” (and their variants) areopen-ended linking verbs and allow the addition of other elements whenused in a claim. “Consisting of” is closed, and “consisting essentiallyof” means that non-material elements can be added, such as backgroundmutations that do not effect the reverse beta oxidation pathway, ordiffering media, buffers, salts, and the like.

As used herein “longer chain” alcohol, fatty acid and the like means 3or more carbons, and preferably a 4 carbon or higher.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A Proposed metabolic platform for the combinatorial synthesis ofadvanced fuels and chemicals using a functional reversal of theβ-oxidation cycle. The engineered reversal of the β-oxidation cyclereported in this work is composed of the following enzymes (gene namesin parentheses): {circle around (1)} thiolase (yqeF, fadA, atoB);{circle around (2)} hydroxyacyl-CoA dehydrogenase (fadB, fadJ); {circlearound (3)} enoyl-CoA hydratase (fadB, fadJ) (note: {circle around (2)}and {circle around (3)} are fused in this species); {circle around (4)}acyl-CoA dehydrogenase (ydiO, fadE). Each turn of the reverse cyclegenerates an acyl-CoA that is two carbons longer than the initialacyl-CoA thioester (indicated as C_(n+2)). Intermediates in theengineered pathway can be converted to a functionally diverse set ofalcohols and carboxylic acids of different chain lengths using one ormore of i) alcohol-forming CoA thioester reductases or ii)aldehyde-forming CoA thioester reductases and alcohol dehydrogenases({circle around (5)}) or iii) CoA thioester hydrolases/thioesterases, oracyl-CoA:acetyl-CoA transferases, or phosphotransacylase and carboxylatekinases ({circle around (6)}), as indicated. Products whose synthesiswas demonstrated in this study are shown in boxes. Abbreviations: Rindicates the side chain (e.g. R═H for acetyl-CoA and R═CH₃ forpropionyl-CoA) attached to the acyl-CoA group of the primer or startermolecule. Dotted lines indicate multiple steps while dashed lineswithout arrowheads connect identical metabolites of different chainlength. A comparison of n-alcohol production between the reversal of theβ-oxidation cycle engineered in this work and the recently proposedfatty acid biosynthesis pathway² is shown in FIG. 4.

FIG. 1B Regulation of operons encoding the proposed reversal of then-oxidation cycle by regulators FadR, ArcAB, FNR, CRP-cAMP, AtoSC.Activation and repression of operons is indicated by “↓” and “⊥”,respectively.

FIG. 2 Engineered one-turn reversal of the n-oxidation cycle for thesynthesis of n-butanol and short-chain carboxylic acids. (a) Effect ofgene overexpressions and knockouts (indicated underneath x axis) on thesynthesis of n-butanol and ethanol in strain RB02 (fadR atoC(c) crp*ΔarcA ΔadhE Δpta ΔfrdA). Experiments were performed at 30° C. for 24hours in shake flasks using glucose (1% w/v) minimal medium. Then-butanol yield was calculated as g n-butanol/g total glucose consumed.(b) Kinetics of n-butanol production by strain RB02 ΔyqhD ΔeutE[yqeF+fucO+]. Cells were cultivated in fermentors containing minimalmedium supplemented with 5% (w/v) glucose. The dissolved oxygen wascontrolled at 5% of saturation, temperature at 30° C., and pH at 7. (c)Synthesis of β-ketobutyric (left panel), β-hydroxybutyric (centerpanel), and trans-2-butenoic (right panel) acids upon overexpression ofthioesterases I (TesA) and II (TesB) in strains RB02, RB02ΔfadB, andRB02ΔydiO. Experiments were run at 37° C. for 48 hours in shake flasksusing glucose (1% w/v) minimal medium.

FIG. 3 Synthesis of higher-chain (C>4) carboxylic acids (a and b) andn-alcohols (c and d) through the engineered reversal of the n-oxidationcycle. (a) Accumulation of long-chain (C>10) free fatty acids in theextracellular medium of strain RB03 [fadBA+] upon overexpression ofdifferent thioesterases (FadM, YciA, TesA, TesB). Product yield is shownabove the bars (g free fatty acid/g total glucose consumed×100).Experiments were run at 37° C. for 48 hours in shake flasks usingglucose (2% w/v) minimal medium. (b) Kinetics of fatty acid synthesis bystrain RB03 [fadBA.fadM+]. Cells were cultivated in fermentors usingglucose (3% w/v) minimal medium. The dissolved oxygen was controlled at2% of saturation, temperature at 37° C., and pH at 7. (c) Synthesis ofn-alcohols in strain RB03 [fadBA+] upon overexpression of alcoholdehydrogenases (YiaY, BetA, and EutG). Product yield is shown above thebars (g n-alcohol/g total glucose consumed×100). Experiments were run at37° C. for 48 hours in shake flasks using glucose (2% w/v) minimalmedium. (d) Effect of alcohol dehydrogenase overexpression (YiaY, BetA,and EutG) on the chain-length distribution of n-alcohols synthesized bystrain RB03 [fadBA+] in the presence of 0.5 g/L propionate. Experimentswere conducted as described in panel “c”.

FIG. 4 is a comparison of n-alcohol synthesis via the fatty acidbiosynthesis pathway (top) and the engineered reversal of theβ-oxidation cycle (bottom). Reactions {circle around (1)}-{circle around(5)} are as indicated in FIG. 1. The fatty acid biosynthesis pathwayuses acyl-ACP intermediates and involves a β-ketoacyl-acyl-carrierprotein synthase ({circle around (8)}) along with a 3-ketoacylreductase, an enoyl reductase, and a 3-hydroxyacyl dehydratase ({circlearound (9)}). The synthesis of malonyl-ACP, the 2-C donor during chainelongation in fatty acid biosynthesis, is also shown ({circle around(7)}). Production of n alcohols from these acyl-ACP intermediatesrequires their conversion to free acids ({circle around (10)}) andacylation ({circle around (11)}) before their reduction to alcohols({circle around (6)}) can be achieved. The use of acyl-ACP intermediatesand malonyl-ACP as the 2-C donor during chain elongation in the fattyacid biosynthesis pathway limits its ATP efficiency, making it anATP-consuming pathway, as shown in the following balanced equation forn-alcohol synthesis from glucose:n/4C₆H₁₂O₆+ATP→C_(n)H_(n+2)O+n/2CO₂+(n/2−1)H₂O,

with n being the chain length of the n-alcohol.

FIG. 5 is synthesis of hydrocarbons through engineered reversal of theβ-oxidation cycle and efficient operation of the core pathway by optimalcoupling of generation and consumption of reducing equivalents.Termination pathways “5” and “6” enable the synthesis of hydrocarbonsfrom CoA-thioester intermediates using aldehyde-forming CoA thioesterreductases and aldehyde decarbonylases (pathway “5”, leading to theformation of alkanes or terminal alkenes of different chain lengths) andolefin-forming enzymes (pathway “6”, leading to the formation ofaliphatic internal alkenes or terminal alkenes or trienes or alkenols).Also noted are dissimilation of pyruvate through routes that preservereducing equivalents (as opposed to releasing them in the form ofhydrogen: e.g. PDH*, PNO, YdbK, NADH-dependent FDH), use ofNAD(P)H-dependent trans-enoyl-CoA reductases (reaction 4), and directcoupling between trans-enoyl-CoA reduction (reaction 4) and pyruvateoxidation.

FIG. 6 is a diagram illustrating the map of plasmid pZS acrMsynpcc7942_1593. This plasmid expresses codon optimized AcinetobacteracrM gene (encoding an aldehyde-forming CoA thioester reductase) andcodon optimized Synechococcus synpcc7942_1593 gene (encoding an aldehydedecarbonylases). These two enzymes form a termination pathway that leadsto the formation of alkanes or terminal alkenes of different chainlengths from the CoA-thioester intermediates of the β-oxidationreversal.

FIG. 7 contains diagrams illustrating the maps of plasmids pZS Egter (A)and pZS Tdter (B), carrying the genes that encode E. gracilis and T.denticola NAD(P)H-dependent transenoyl-CoA dehydrogenases, respectively.

FIG. 8 contains diagrams illustrating the maps of plasmids pZS ydbK (A)pZS ydbKydiQRST (B), and pZS ydiO (C). These plasmids carry the genesthat code for E. coli pyruvate:flavodoxin oxidoreductase (YdbK),acyl-CoA dehydrogenase (YdiO) and required electron transferflavoproteins and ferredoxin (YdiQRST).

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

We have engineered a functional reversal of the fatty acid oxidationcycle (aka β-oxidation) in E. coli and used it in combination withendogenous dehydrogenases and thioesterases to produce n-alcohols andfatty acids of different chain lengths (FIG. 1).

The engineered pathway operates with coenzyme-A (CoA) thioesterintermediates and uses acetyl-CoA directly for acyl-chain elongation(rather than first requiring ATP-dependent activation to malonyl-CoA),features that enable product synthesis at maximum carbon and energyefficiency.

The synthesis of substituted and ubsubstituted n-alcohols and carboxylicacids (FIGS. 2 and 3) at yields and titers higher than previouslyreported demonstrate the superior nature of the engineered pathway. Theubiquitous nature of the n-oxidation cycle should enable the efficientsynthesis of a host of non-native products in industrial organismswithout recruiting foreign genes, an approach we term here homologousmetabolic engineering.

We will shortly demonstrate the production of alkanes and alkenes usingnon-native enzymes added to the bacteria, and the vectors for same havealready been constructed.

EXAMPLE 1 Materials and Methods

The material and methods detailed herein are exemplary only, but thetechniques are standard in the art and different methodologies can besubstituted herein. What is important is the engineering to effectpathway reversal, direct carbon flow, and upregulating the terminationenyzmes.

Reagents

Chemicals were obtained from FISHER SCIENTIFIC™ (Pittsburgh, Pa.) andSIGMA-ALDRICH CO.™ (St. Louis, Mo.).

Culture Medium

The minimal medium designed by Neidhardt (1974) with Na₂HPO₄ in place ofK₂HPO₄ and supplemented with 20 g/L glucose, 40 g/L calcium bicarbonate,100 μM FeSO₄, 5 mM calcium pantothenate, 3.96 mM Na₂HPO₄, 5 mM(NH₄)₂SO₄, and 30 mM NH₄Cl was used. Fermentations conducted in theSIXFORS™ multi-fermentation system also included 1 mM betaine.

Plasmid Construction

Standard recombinant DNA procedures were used for gene cloning, plasmidisolation, and electroporation. Manufacturer protocols and standardmethods were followed for DNA purification (QIAGEN,™ CA, USA),restriction endonuclease digestion (NEW ENGLAND BIOLABS,™ MA, USA), andDNA amplification (STRATAGENE,™ CA, USA and INVITROGEN,™ CA, USA). Forplasmid construction, genes were amplified from MG1655 genomic DNA usingprimers designed to create 15 bp of homology on each end of the geneinsert for subsequent recombination into the desired plasmid. Plasmidswere linearized using restriction endonuclease digestion, thenrecombined with the appropriate gene(s) using an IN-FUSION DRY-DOWN PCRCLONING KIT™ (CLONTECH,™ Mountain View, Calif., USA) and subsequentlyused to transform chemically competent FUSION BLUE™ cells (CLONTECH,™Mountain View, Calif., USA).

Transformants that grew on LB plates containing the appropriateantibiotic were struck for isolation, and then subjected to preliminaryscreening by PCR. Colonies passing preliminary inspection were thenindividually grown for plasmid purification. Purified plasmids wereconfirmed to have the appropriate insert both by PCR as well asrestriction endonuclease digest verification. Plasmids in each caseinclude the plasmid promoter, a ribosomal binding site for each gene,MG1655 gene(s), and a plasmid terminator. Resulting plasmids (andstrains) are listed in Tables 3 and 4.

Metabolite Identification

The identity of metabolic products was determined throughone-dimensional (1D) proton nuclear magnetic resonance (NMR)spectroscopy. 60 μL of D₂O and 1 μL of 600 mM NMR internal standard TSP[3-(trimethylsilyl) propionic acid-D4, sodium salt] were added to 540 μLof the sample (culture supernatant). The resulting solution was thentransferred to a 5 mm-NMR tube, and 1D proton NMR spectroscopy wasperformed at 25° C. in a Varian 500-MHz Inova spectrometer equipped witha Penta probe (VARIAN, INC.,™ Palo Alto, Calif.) using the followingparameters: 8,000-Hz sweep width, 2.8-s acquisition time, 256acquisitions, 6.3-μs pulse width, 1.2-s pulse repetition delay, andpresaturation for 2 s. The resulting spectrum was analyzed using FELIX™2001 software (ACCELRYS SOFTWARE INC.,™ Burlington, Mass.). Peaks wereidentified by their chemical shifts and J-coupling values, which wereobtained in separate experiments in which samples were spiked withmetabolite standards (2 mM final concentration).

Identification of n-alcohols was conducted through gaschromatography-mass spectroscopy (GC-MS) following a modification of themethod reported by Atsumi (2008). The analysis was performed on anAGILENT™ 6890 GC/5973 MS (AGILENT TECHNOLOGIES,™ Palo Alto, Calif.)instrument with a HP-5 ms capillary column (30 m×0.25 mm×0.25 μm). 1 mlof supernatant of culture broth was extracted with 500 μl of GC standardgrade hexane (Fluka). 0.5 μl of the extracted sample was injected usinga 20:1 split at 250° C. The oven temperature was initially held at 75°C. for 2 min and then raised with a gradient of 5° C./min to 280° C. andheld for 2 min. Helium (MATHESON TRI-GAS,™ Longmont, Colo.) was used asthe carrier gas with a 14-lb/in² inlet pressure. The injector anddetector were maintained at 255° C.

Identification of fatty acids was performed on a SHIMADZU™ Auto-SystemGC 2010 (SHIMADZU,™ Japan) equipped with a DB-5MS capillary column (30m×0.25 mm×0.25 μm) and directly connected to MS. The following methodwas used: an initial temperature of 50° C. was held for 2 min and thenramped to 220° C. at 4° C. per min and held for 10 min². Extraction andderivatization procedures are described in section MetaboliteQuantification.

Metabolite Quantification

The quantification of glucose, organic acids, ethanol, and butanol wasconducted by high-performance liquid chromatography (HPLC). Samples(culture supernatant) were analyzed with ion-exclusion HPLC using aSHIMADZU™ Prominence SIL 20 system (SHIMADZU SCIENTIFIC INSTRUMENTS,INC.,™ Columbia, Md.) equipped with an HPX-87H organic acid column(BIO-RAD,™ Hercules, Calif.) with operating conditions to optimize peakseparation (0.3 mL/min flowrate, 30 mM H₂SO₄ mobile phase, columntemperature 42° C.).

Quantification of longer chain (C>4) n-alcohols was conducted throughgas chromatography (GC) in a VARIAN™ CP-3800 gas chromatograph (VARIANASSOCIATES, INC.,™ Palo Alto, Calif.) equipped with a flame ionizationdetector (GC-FID). Sample extraction procedure was as described above insection Metabolite Identification. The separation of alcohol compoundswas carried out using a VF-5ht column (15 m, 0.32 mm internal diameter,0.10 μm film thickness; VARIAN ASSOCIATES, INC.,™ Palo Alto, Calif.).The oven temperature was initially held at 40° C. for 1 min and thenraised with a gradient of 30° C./min to 130° C. and held for 4 min. Thetemperature was then raised with a gradient of 15° C./min to 230° C. andheld for 4 min. Helium (1 ml min⁻¹, MATHESON TRI-GAS,™ Longmont, Colo.)was used as the carrier gas. The injector and detector were maintainedat 250° C. A 0.5-μl sample was injected in splitless injection mode.

Quantification of fatty acids was carried out in a VARIAN™ CP-3800 gaschromatograph (VARIAN ASSOCIATES, INC.,™ Palo Alto, Calif.) afterhexane-methyl tertiary butyl ether (MTBE) extraction (Lalman 2004) andFA transesterification with a mixture ofcholophorm:methanol:hydrochloric acid [10:1:1, vol/vol/vol] aspreviously reported (Dellomonaco 2010). The resulting fatty acids methylesters were quantified according to the following method: 50° C. heldfor 1 min, 30° C./min to 160° C., 15° C./min to 200° C., 200° C. heldfor 1.5 min, 10° C./min to 225° C., and 225° C. held for 15 min.

Enzyme Assays

For measurement of enzymatic activities, cells from 24 hour shake flaskcultures were washed twice with 9 g/L sodium chloride under anaerobicconditions and stored at −80° C. until use. Cell extracts for all assayswere prepared as follows under anaerobic conditions. 40 units ofOD_(550 nm) was re-suspended in 1 mL of 100 mM Tris-HCl buffer (pH 7.0)with 1 mM DTT. After cellular disruption using a DISRUPTOR GENIE™(SCIENTIFIC INDUSTRIES, INC.,™ Bohemia, N.Y.), cellular debris wasremoved by centrifugation (13,000×g, 4° C., 10 min) and the supernatantused as cell extract. Absorbance changes for all assays were monitoredin a BIOMATE™ 5 spectrophotometer (THERMO SCIENTIFIC,™ MA, USA). Thelinearity of reactions (protein concentration and time) was establishedfor all assays and the non-enzymatic rates were subtracted from theobserved initial reaction rates. Enzymatic activities are reported asμmol of substrate per minute per mg of cell protein and representaverages for at least three cell preparations. Protein concentration wasmeasured using the Bradford assay reagent (THERMO SCIENTIFIC,™ MA, USA)with BSA as a standard.

Acetyl-CoA acetyltransferase (THL) activity was determined usingacetoacetyl-CoA and CoA as substrates, and the decrease inacetoacetyl-CoA concentration was measured at 303 nm.β-Hydroxybutyryl-CoA dehydrogenase activity was measured at 340 nm bymonitoring the decrease in NADH concentration resulting fromβ-hydroxybutyryl-CoA formation from acetoacetyl-CoA. Crotonase activitywas measured by monitoring the decrease in crotonyl-CoA concentration at263 nm, which results from β-hydroxybutyryl-CoA formation fromcrotonyl-CoA. Butyryl-CoA dehydrogenase activity was assayed in thedirection of crotonyl-CoA reduction by monitoring the ferricenium ion at300 nm, which acts as an electron donor. In addition, assays in whichthe ferricenium ion was replaced with 0.2 mM NAD(P)H and the absorbancemeasured at 340 nm were also run. Butyraldehyde dehydrogenase activitywas assayed in the direction of butyraldehyde oxidation by monitoringNAD(P)⁺ reduction at 340 nm. To measure butanol dehydrogenase activity,the decrease in NAD(P)H concentration resulting from butanol formationfrom butyraldehyde is monitored at 340 nm under anaerobic conditions at30° C.

EXAMPLE 2 One-Turn Reversal of β-Oxidation Cycle

Given the applications of n-butanol as both advanced biofuels andbuilding blocks for the chemical industry, we chose it as the firstproduct to demonstrate the feasibility of engineering a functionalreversal of the β-oxidation cycle as an efficient platform for fuel andchemical production (FIG. 1).

Synthesis of n-butanol can be realized through a one-turn reversal ofthe β-oxidation cycle in combination with native aldehyde/alcoholdehydrogenases (FIG. 1a , reactions {circle around (1)}-{circle around(5)}). This engineered pathway represents an E. coli surrogate of then-butanol pathway operating in Clostridia.

Given the specificity of atoB-encoded acetyl-CoA acetyltransferase forshort-chain acyl-CoA molecules and the high sequence similarity betweenatoB and yqeF (predicted acyltransferase), these genes were selected forReaction {circle around (1)} of the pathway. The next two steps can becatalyzed by 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases,encoded by fadB and fadJ (Reactions {circle around (2)} and {circlearound (3)} in FIG. 1a ). The fourth step in this one-turn reversal ofthe β-oxidation cycle can be catalyzed by acyl-CoA dehydrogenase (fadEor ydiO) (Reaction {circle around (4)}).

The above genes are organized in four operons in the E. coli genome andare subjected to several levels of regulation (FIG. 1b ). Theseregulatory pathways were therefore engineered to promote the reversal ofthe n-oxidation cycle.

Constitutive expression of fad and ato genes (regulated by FadR andAtoC, respectively: FIG. 1b ) was achieved through fadR and atoC(c)mutations (Dellomonaco 2010). Since anaerobic/microaerobic conditionsused in the production of fuels and chemicals would lead to repressionof most target operons by ArcA (FIG. 1b ), the arcA gene was alsodeleted.

Several operons of interest are also activated by the cyclic-AMPreceptor protein (CRP)-cAMP complex (FIG. 1b ) and are thereforesubjected to carbon catabolite repression in the presence of glucose.This regulatory mechanism was circumvented by replacing the native crpgene with cAMP-independent mutant crp* (Eppler & Boos, 1999). Whilethese genetic manipulations were predicted to enable expression of theβ-oxidation cycle (FIG. 1b ), no butanol synthesis was observed instrain fadR atoC(c) Δcrp crp* ΔarcA (RB01) or its parent fadR atoC(c)(Table 2). Table 5 provides details about mutations introduced at thecrp, fadR, and atoSC loci.

Given the significant accumulation of other fermentation products (Table2), the pathways involved in the synthesis of ethanol, acetate, andsuccinate were also blocked (ΔadhE, Δpta and ΔfrdA knockouts,respectively) in an attempt to channel carbon to the engineered pathway.Although the synthesis of these competing by-products was greatlyreduced, strain RB02 (RB01 ΔadhE Δpta ΔfrdA) did not produce n-butanoleither (Table 2).

Enzyme activity measurements confirmed a functional expression of thereversal of the β-oxidation cycle in strain RB02, compared to negligibleactivity in wild-type MG1655 (Table 1a). However, the levels ofn-butanol dehydrogenase were very low (Table 1a), probably preventingn-butanol synthesis.

To address this issue, two endogenous aldehyde/alcohol dehydrogenaseswith high sequence and structure similarity to the Clostridialbutyraldehyde/butanol dehydrogenase were overexpressed in strain RB02:i.e. L-1,2-propanediol oxidoreductase (fucO) and an aldehyde/alcoholdehydrogenase (yqhD) (Table 7). Despite the potential for YqhD tocatalyze the conversion of butyraldehyde to n-butanol, overexpression offucO led to higher n-butanol titer and yield (FIG. 2a ). Nonetheless,both enzymes proved functional and either or both could be used.

Although high levels of thiolase activity were observed in RB02 (Table1a), these measurements account for enzymes with specificity for bothshort- and long-chain acyl-CoA molecules. In an attempt to divertacetyl-CoA to the n-butanol pathway, acetyl-CoA acetyltransferases thatpossess higher affinity for short-chain molecules (atoB and yqeF: FIG.1a , Reaction {circle around (1)}) were overexpressed. The resultingstrains, RB02 [atoB+] and RB02 [yqeF+], synthesized appreciable amountsof n-butanol (FIG. 2a ).

Overexpression of yqeF, whose function in E. coli metabolism iscurrently unknown, yielded higher concentrations of n-butanol and lowerconcentrations of the major fermentation by-product ethanol (FIG. 2a ).No n-butanol synthesis was observed upon overexpression of atoB or yqeFin wild-type MG1655.

Based on the above results, an increased partition of carbon fluxtowards n-butanol should be realized by simultaneous overexpression ofyqeF, to channel acetyl-CoA into the engineered reversal of theβ-oxidation cycle, and fucO, to improve the conversion of butyryl-CoA ton-butanol. Indeed, strain RB02 [yqeF+fucO+] produced significant amountsof n-butanol (1.9 g/L) at a high n-butanol-to-ethanol ratio (>5:1) (FIG.2a ). No n-butanol production was observed upon simultaneousoverexpression of yqeF and fucO in the wild-type background or in astrain containing pta, adhE, and frdA deletions, underscoring theimportance of the fadR atoC(c) Δcrp crp* ΔarcA genotype.

Since the engineering of strain RB02 [yqeF+fucO+] involved manipulationof several global regulators with potential pleiotropic effects, acharacterization of the proposed reversal of the β-oxidation cycle wasconducted to establish its role on n-butanol synthesis (Table 1).Activity measurements showed high level of expression of key enzymesinvolved in the postulated pathway in strain RB02 [yqeF+fucO+] andnegligible activity in wild type MG1655 (Table 1a). Gene knockout andgene complementation experiments along with quantification offermentation products (Table 1b) demonstrated that the primary genesinvolved in the synthesis of n-butanol through the engineered one-turnreversal of the β-oxidation pathway are (encoded activity inparenthesis): yqeF (predicted acyltransferase), fadB (3-hydroxyacyl-CoAdehydrogenase and enoyl-CoA hydratase), ydiO (predicted acyl-CoAdehydrogenase), and fucO (L-1,2-propanediol oxidoreductase/n-butanoldehydrogenase).

YdiO is proposed to catalyze the reduction of enoyl-CoA to acyl-CoA(Reaction {circle around (4)}). The reverse of this reaction iscatalyzed by FadE and is the only irreversible step in the catabolicoperation of the β-oxidation cycle⁵. In agreement with our proposal,deletion of ydiO in strain RB02 [yqeF+fucO+] completely abolishedn-butanol synthesis (Table 1b). Although ydiO was previously proposed toencode an acyl-CoA dehydrogenase that would replace FadE during theanaerobic catabolism of fatty acids²⁰, a sequence comparison betweenYdiO and E. coli proteins does not reveal a significant similarity toFadE (Table 9). In contrast, YdiO shares high homology withcrotonobetainyl-CoA reductase (CaiA). CaiA catalyzes the reduction ofcrotonobetainyl-CoA to γ-butyrobetainyl-CoA, a reaction similar to thatcatalyzed by YdiO in the reversal of the β-oxidation cycle. Moreover,the operon fixABCX is required for the transfer of electrons to CaiA andencodes flavoproteins and ferredoxin with high sequence similarity toYdiQRST (Table 9). This analysis suggests that ferredoxin andflavoproteins encoded by ydiQRST are involved in the transfer ofelectrons to YdiO during the reduction of enoyl-CoA to acyl-CoA.Standard Gibbs energy calculations revealed that the engineered reversalof the β-oxidation cycle is thermodynamically feasible if ferredoxin isthe source of reducing power for the conversion of enoyl-CoA to acyl-CoA(Table 10). We then propose that the reduction of enoyl-CoA to acyl-CoAis mediated by YdiO-YdiQRST.

Further reduction in the synthesis of by-product ethanol, and hence anincrease in n-butanol yield, were realized by combining theoverexpression of fucO and yqeF with the deletion of yqhD and eutE(aldehyde dehydrogenase with high sequence similarity to adhE). Theresulting strain (RB02 ΔyqhD ΔeutE [yqeF+fucO+]) synthesized 2.2 g/L ofn-butanol in 24 hours at a yield of 0.28 g n-butanol/g glucose (FIG. 2a). When grown in a bioreactor using a higher initial concentration ofglucose, this strain produced n-butanol at high titer (˜14 g/L), yield(0.33 g n-butanol/g glucose) and rate (˜2 g n-butanol/g cell dryweight/h) (FIG. 2b ).

This performance, which was achieved without importing foreign genes andin the absence of rich nutrients, is an order of magnitude better thanreported for any other organism engineered for n-butanol production andalso surpasses the yield and specific productivity reported for nativen-butanol producers. The reversal of the β-oxidation cycle engineered inthis strain operated at a maximum carbon flux of 73.4 mmol acetyl-CoA/gcell dry weight/h (12-18 hours in FIG. 2b ), which exceeds the fluxreported in the literature for native or engineered fermentativepathways. Taken together, these results demonstrate that the engineeredreversal of the β-oxidation pathway is a superior metabolic platform forthe production of fuels and chemicals and can support the efficientsynthesis of non-native products in industrial organisms withoutrecruiting foreign genes (i.e. endogenous metabolic engineering).

The engineered reversal of the β-oxidation cycle generates a diverse setof CoA thioester intermediates that can be converted to thecorresponding alcohols and carboxylic acids (FIG. 1A). To illustrateproduct synthesis from intermediates other than acyl-CoA, we usedthioesterase I (TesA) and thioesterase II (TesB) as termination enzymes.Small amounts of 3-hydroxybutyric, 3-ketobutyric, and trans-2-butenoicacids were produced when these thioesterases were overexpressed instrain RB02 (FIG. 2c ). The level of these products was significantlyincreased by simultaneous overexpression of thioesterase andyqeF-encoded short-chain acyltransferase (FIG. 2c ). Further increasesin product titer were realized upon deletion of fadB (˜500 mg/L3-ketobutyric acid) and ydiO (˜150 mg/L and 200 mg/L of 3-hydroxybutyricand trans-2-butenoic acids, respectively) (FIG. 2c ).

EXAMPLE 3 Making Longer Chains (C>4)

The operation of multiple cycles of the engineered reversal of theβ-oxidation pathway, and hence the synthesis of CoA-thioesterintermediates (and products) of longer chain length (C>4), can befacilitated by the overexpression of FadA, a 3-ketoacyl-CoA thiolasethat is part of the β-oxidation complex (FadBA) and which possessesbroad chain length specificity.

Overexpression of FadBA in conjuction with thioesterases (TesA, TesB,FadM or YciA) in strain RB03 (RB02ΔyqhDΔfucO ΔfadD) resulted in theaccumulation of long-chain fatty acids in the extracellular medium (FIG.3a ). The fadD knockout in strain RB03 prevents re-utilization of thesynthesized fatty acids. The choice of thioesterase allowed control overboth length and functionality of the fatty acid side chain. For example,C16 and C18 saturated fatty acids were the only products when FadM wasoverexpressed while YciA and TesA overexpression supported the synthesisof 3-hydroxy (C14:3OH) and unsaturated (C18:1) fatty acids, respectively(FIG. 3a ).

When grown in a bioreactor using a higher initial concentration ofglucose, strain RB03 [fadBA.fadM+] produced long-chain extracellularfatty acids at high titer (˜7 g/L) and yield (0.28 g fatty acids/g totalglucose consumed) using a mineral salts medium without rich nutrients(FIG. 3b ). These results are better than reported previously using anengineered fatty acid biosynthesis pathway (Table 9). No production ofextracellular free fatty acids was observed upon overexpression of FadMin strain MG1655 ΔadhE Δpta ΔfrdA ΔfadD (Table 6C), demonstrating therequirement of an active reversal of the β-oxidation cycle. Measurementsof total free fatty acids (i.e. extracellular+intracellular) in strainRB03 [fadBA.fadM+] and the corresponding controls showed that theengineered reversal contributed to the synthesis of 90-95% of the totalfree fatty acids (Table 6C).

The synthesis of longer-chain (C>4) n-alcohols was also demonstrated byoverexpressing the appropriate termination enzymes (FIG. 3c ). Weidentified native enzymes that could serve as potential surrogates forthe aldehyde-forming acyl-CoA reductases and alcohol dehydrogenasespresent in organisms that synthesize higher-chain linear n-alcohols(Table 7). The product titer (0.33 g/L) and yield (8.3% w/w) achievedupon overexpression of YiaY were higher than previously reported (Table8).

Synthesis of odd-chain n-alcohols was demonstrated by supplementing themedium with propionate as the precursor of propionyl-CoA (R═CH₃ in FIG.1A). A clear shift in the distribution of n-alcohols was observed:odd-chain alcohols 1-pentanol, 1-heptanol, and 1-nonanol appeared asfermentation products and the synthesis of even-chain alcoholssignificantly decreased (FIG. 3d ).

EXAMPLE 4 Synthesis of Alkanes/Alkenes

The CoA-thioester intermediates generated by the reversal of theβ-oxidation cycle can be converted to alkanes by a two-step pathwaycomposed of an aldehyde-forming fatty-AcylCoA reductase and a fattyaldehyde decarbonylase (FIG. 5). Olefins, on the other hand, will alsobe synthesized from acyl-CoAs via a pathway that uses a “head-to-head”condensation mechanism followed by reduction and decarbonylation steps.

Alkane-Biosynthesis Pathway:

A two-step pathway will be used, which involves the reduction ofacyl-CoA to fatty aldehydes by the action of fatty aldehyde-formingacyl-CoA reductases followed by the decarbonylation of the resultingaldehyde to alkane by aldehyde decarbonylases (FIG. 5). This pathway isdifferent from a recently reported pathway for the synthesis of alkanesfrom fatty-acyl-ACP (acyl-acyl carrier protein). Our pathway uses anacyl-CoA reductase as opposed to the reported acyl-ACP reductase. Wehave already used native fatty aldehyde-forming acyl-CoA reductases inthe synthesis of fatty alcohols. In addition, we will use heterologousfatty aldehyde-forming acyl-CoA reductases from Acinetobactercalcoaceticus (acr1) and Acinetobacter sp. strain M-1 (acrM) (Ishige etal., 2002).

While both enzymes are active with a range of acyl-CoAs, the activitytowards palmitoyl-CoA is very high: this is an important aspect becauseour strains engineered to produce fatty acids synthesize palmitic acidas the primary product (FIG. 3b ), indicating the availability ofpalmitoyl-CoA for the fatty aldehyde-forming acyl-CoA reductases.

For the second step of the pathway we will use an aldehyde decarbonylasefrom Synechococcus elongatus PCC7942 (PCC7942_orf1593) and otherorthologs recently reported by Schirmer (2010).

Genes encoding the aforementioned enzymes were clustered in the sameexpression vector (FIG. 6), which will be transformed into strains thatwe have already shown to be able to produce long-chain fatty acids fromacyl-CoAs. Heterologous genes were codon-optimized for expression in E.coli. The effect of the expression levels of each enzyme in the pathwaywill be assessed through the use of different expression vectors,promoters and ribosomal binding sites, etc. Once alkane production hasbeen verified, the vector carrying the alkane-biosynthesis pathway (FIG.6) will be expressed in conjunction with a second vector carrying theβ-oxidation enzymes.

The activity of proteins encoded by cloned genes will be quantified andthe corresponding reactions characterized using in vitro analysis ofenzyme kinetics and identification of reaction substrates and productsusing biochemical assays and NMR spectroscopy. Substrates with differentchain length will be used in these assays.

Olefin-Biosynthesis Pathway:

The best-characterized pathway for the synthesis of olefins proceedsthrough a mechanism known as “head-to-head” condensation of acyl-CoAsand leads to the synthesis of long-chain olefins (C21-C31) with internaldouble bonds at the median carbon.

The optimal functioning of this pathway will require the expression ofthe cluster of olefin-forming enzymes OleABCD from bacteria such asXanthomonas campestris. Recent in vitro studies have shown that OleAcatalyzes the condensation of fatty acyl groups in the first step of thepathway through a non-decarboxylative Claisen condensation mechanism.Purified OleA was shown to be active with fatty acyl-CoAs that rangedfrom C8 to C16 in length, with maximum activity towards palmitoyl-CoA.The other three genes encode a member of the α/β-hydrolase superfamily(OleB), a member of the AMPdependent ligase/synthase superfamily oracetyl-CoA synthetase-like superfamily (OleC), and a member of theshort-chain dehydrogenase/reductase superfamily (OleD).

The genes acyl, acrM, PCC7942 orf1593, oleABCD will be cloned in oneexpression vector and the effect of the expression levels of each enzymein the pathway will be assessed through the use of different promotersand ribosomal binding sites, as described above. A second vector will beused to express the β-oxidation enzymes. The vectors will be transformedinto strains already shown to be able to produce long-chain fatty acidsfrom acyl-CoAs. The activity of proteins encoded by the cloned geneswill be quantified and the corresponding reactions characterized usingin vitro analysis of enzyme kinetics and identification of reactionsubstrates and products using biochemical assays and NMR spectroscopy.

EXAMPLE 5 Improving Efficiency of Reversed Cycle

The synthesis and consumption of reducing equivalents is a key aspectfor the efficient operation of the engineered pathway, we propose toimprove its functioning by manipulating the enzymes responsible fortrans-enoyl-CoA reduction and pyruvate oxidation (FIG. 1A and FIG. 5).This includes dissimilation of pyruvate through routes that preservereducing equivalents (as opposed to releasing them in the form ofhydrogen), use of NAD(P)H-dependent trans-enoyl-CoA reductases, anddirect coupling between trans-enoyl-CoA reduction and pyruvateoxidation.

Pyruvate can be converted to acetyl-CoA in E. coli through three mainroutes (FIG. 5): i) pyruvate formate-lyase (PFL), which generatesformate as co-product, ii) pyruvate dehydrogenase (PDH), which generatesCO₂ and NADH, and iii) a YdbK, a predicted pyruvate:flavodoxinoxidoreductase, that also generates CO₂ and transfer the electrons tothe quinone pool. While the formate generated by PFL can bedisproportionated to CO₂ and hydrogen by the action of formatehydrogenlyase (FHL), this enzyme does not generate NAD(P)H.

To address this issue, we will replace the native hydrogen-evolving FHLcomplex with an NAD-dependent formate dehydrogenase (FDH) from C.boidinii (FIG. 5). PDH and YdbK generate reducing equivalents in a formpotentially usable by the engineered reversal of the b-oxidationpathway. However, effective functioning of PDH would require the use ofanaerobic conditions or the replacement of the native PDH with ananaerobically active pyruvate dehydrogenase complex (PDH*) (Kim et al.,2007). In the case of YdbK, we propose the direct coupling of pyruvateoxidation and YdiO-catalyzed reduction of trans-enoyl-CoA. We will alsoevaluate the expression of a heterologous pyruvate-NADP oxidoreductase(PNO) from the mitochondrion of Euglena gracilis and from theapicomplexan Cryptosporidium parvum, which convert pyruvate toacetyl-CoA, CO₂, and NADPH (Rotte et al., 2001).

Two enzymes will be evaluated for the reduction of trans-enoyl-CoA,namely NAD(P)H-dependent trans-enoyl-CoA reductase from Euglena gracilis(Hoffmeister et al., 2005) and predicted E. coli acyl-CoA dehydrogenase(YdiO). In the case of YdiO, we have recently shown that this enzyme isrequired for the operation of the reversal of the b-oxidation pathway(Dellomonaco et al., 2011). The effect of availability of reducingequivalents in the form of NADPH or NADH will also be evaluated throughmanipulation of the flux through transhydrogenases as well as the carbonflux partitioning between Embden-Meyerhof-Parnas pathway, PentosePhosphate pathway, and the Entner-Doudoroff pathway.

Genes encoding some of the aforementioned enzymes have been cloned inappropriate expression vectors (FIG. 7 and FIG. 8) and will betransformed into strains able to produce alcohols, carboxylic acids,alkanes, and alkenes via a an engineered reversal of the β-oxidationcycle. The encoded activities and corresponding reactions will becharacterized using in vitro analysis of enzyme kinetics andidentification of reaction substrates and products using biochemicalassays and NMR spectroscopy.

Conclusions:

The functional reversal of the β-oxidation cycle engineered in this workrepresents a new and highly efficient platform for the synthesis ofadvanced fuels and chemicals. Its superior nature is illustrated in thefollowing balanced equation for the synthesis of n-alcohols fromglucose: n/4C₆H₁₂O₆→C_(n)H_(n+2)O+n/2CO₂+(n/2−1)H₂O+n/2 ATP, with nbeing the chain length of the n-alcohol (FIG. 1a ). As can be seen, theengineered pathway has the potential to achieve the maximum yield ofn-alcohols on glucose (66.7%, C-mole basis) and generates 1 ATP per each2-C incorporated into the n-alcohol molecule. This ATP yield isequivalent to that of efficient homo-fermentative pathways found innature such as ethanol and lactic acid fermentations. The high carbonand energy efficiency of the engineered reversal of the β-oxidationcycle is possible because it uses acetyl-CoA directly as C2 donor duringchain elongation (as opposed to first requiring ATP-dependent activationto malonyl-CoA) and it functions with acyl-CoA intermediates, which arethe precursors of alcohols and other important products (FIG. 1a ).

The synthesis of n-alcohols through alternative metabolic routes, suchas the fatty acid biosynthesis and keto-acid pathways, is lessefficient. For example, the use of the fatty acid biosynthesis pathwayresults in the net consumption of 1 ATP per molecule of n-alcoholsynthesized (FIG. 4). This inefficiency is due to the consumption of ATPin the synthesis of malonyl-ACP (reaction {circle around (7)} in FIG.4), the C2 donor for chain elongation, and the use of acyl-ACPintermediates, which need to be converted to free acids and acylated(another ATP-consuming step) before their reduction to alcohols can beachieved (reactions {circle around (10)} and {circle around (11)} inFIG. 4). The recently proposed keto-acid pathway is also less efficientthan the reversal of the β-oxidation cycle: e.g. the maximum theoreticalyield of n-hexanol, the highest-chain linear n-alcohol reported with theketo-acid pathway, is only 50% C-mole (2Glucose→n-hexanol+ATP+2[H]+6CO₂).

While the work reported here focused on the engineering of E. coli, theubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase, andthioesterase enzymes will certainly enable the use of native metabolicengineering strategies to achieve the efficient synthesis of β-alcoholsand fatty acids in other industrial organisms. A functional reversal ofthe β-oxidation cycle also holds great promise for the combinatorialbiosynthesis of a wide range of molecules of various chain lengths andfunctionalities. For example, thioesterases and aldehyde/alcoholdehydrogenases can also act on the other thioester intermediates of theengineered pathway to generate a host of products such as β-keto acidsand β-keto alcohols, β-hydroxy acids and 1,3-diols, and trans-Δ²-fattyacids and trans-Δ²-alcohols (FIG. 1a ), as well as alkanes and alkenes(FIG. 5).

TABLE 1 Functional characterization of the engineered reversal of theβ-oxidation cycle during the synthesis of n-butanol Table 1a. Activitiesof β-oxidation and butanol dehydrogenase enzymes in wild-type andengineered strains Enzyme activity (μmol/mg protein/min) ± standarddeviation Strain THL^(a) HBD^(a) CRT^(a) BDH^(a) MG1655 n.d. 0.002 ±0.000 n.d. 0.014 ± 0.001 RB02^(b) 0.310 ± 0.079 0.304 ± 0.032 0.339 ±0.049 0.004 ± 0.002 RB02 [yqeF+ fucO+] 0.498 ± 0.036 0.292 ± 0.013 0.334± 0.017 0.298 ± 0.020 Table 1b. Butanol synthesis, glucose utilization,and cell growth in strain RB02 and its derivatives^(c) Butanol producedGlucose Cell Yield (g/g) utilized growth Strain^(d) Concentration (g/L)(g/L) (g/L) RB02 [yqeF+ fucO+] 0.182 1.85 10.19 0.72 Reaction {circlearound (1)}: yqeF (predicted acyltransferase) RB02 ΔyqeF [fucO+] 0.0190.10 5.19 0.32 RB02 [fucO+] 0.063 0.23 3.66 0.41 RB02 ΔyqeF [fucO+yqeF+] 0.159 1.11 7.00 0.43 Reactions {circle around (2)} and {circlearound (3)}: fadB (3-hydroxyacyl-CoA dehydrogenase and enoyl-CoAhydratase) RB02 ΔfadB [yqeF+ fucO+] 0.000 0.00 2.96 0.19 RB02 ΔfadB[yqeF+ fadB+] 0.157 0.16 5.86 0.52 RB02 ΔfadB [yqeF+] 0.000 0.00 1.840.11 Reaction {circle around (4)}: ydiO (predicted acyl-CoAdehydrogenase) RB02 ΔydiO [yqeF+ fucO+] 0.038 0.04 3.14 0.18 RB02 ΔydiO[yqeF+ ydiO+] 0.163 0.16 5.94 0.57 RB02 ΔydiO [yqeF+] 0.018 0.02 1.660.13 Reaction {circle around (5)}: fucO (L-1,2-propanedioloxidoreductase/n-butanol dehydrogenase) RB02 ΔfucO [yqeF+] 0.040 0.042.75 0.38 RB02 ΔfucO [yqeF+ fucO+] 0.151 0.11 5.64 0.51 RB02 [yqeF+]0.088 0.35 4.00 0.68 ^(a)THL: thiolase; HBD: hydroxy-acyl-CoAdehydrogenase; CRT: crotonase; BDH: butanol dehydrogenase; n.d.: notdetected. ^(b)The genotype of strain RB02 is as follows: fadR atoC(c)crp* ΔarcA ΔptaΔadhEΔfrdA. ^(c)Experiments were run for 24 hours inshake flasks using glucose (1% w/v) minimal medium. ^(d)Strains weregrouped based on the relevance of their genotypes for specifiedreactions (see FIG. 1a).

TABLE 2 Cell growth, glucose utilization, product synthesis, and carbonrecovery for wild- type and engineered strains grown on glucose minimalmedium Table 2A. n-butanol synthesis in wild-type and engineered E. colistrains Concentration (g/L) Glucose Strain Cells utilized Butanol %C-recovery^(b) MG1655 0.85 7.33 0.00 88.77 fadR atoC(c) 0.71 5.16 0.0085.78 fadR atoC(c) ΔarcA Δcrp crp* (RB01) 0.53 6.43 0.00 90.23 RB01ΔadhE ΔfrdA Δpta (RB02) 0.29 1.76 0.00 94.09 RB02 [yqhD+] 0.49 4.27 0.1191.70 RB02 [fucO+] 0.41 3.66 0.23 98.48 RB02 [yqeF+] 0.68 4.00 0.3597.13 RB02 [yqeF+ fucO+] 0.72 10.19 1.85 86.05 RB02 ΔyqhD [yqeF+ fucO+]0.49 7.89 1.90 84.15 RB02 ΔyqhD ΔeutE [yqeF+ fucO+] 0.66 7.66 2.15 93.54Table 2B. Synthesis of higher chain (C > 4) n-alcohols by derivatives ofstrain RB03 (RB02 ΔyqhD ΔfucO) Concentration (g/L) Glucose % C- StrainCells utilized n-C6—OH n-C8—OH n-C10—OH recovery^(b) RB03 ΔfadD [fadBA+]0.71 5.27 0.000 0.000 0.000 90.38 RB03 [fadBA+ yiaY+] 0.87 7.19 0.0810.035 0.130 72.71 RB03 ΔfadD [fadBA+ 0.73 5.06 0.170 0.080 0.170 91.97yiaY+] RB03 [fadBA+ eutG+] 0.73 4.68 0.170 0.040 0.000 97.50 RB03 ΔfadD[fadBA+ 0.52 4.00 0.170 0.070 0.010 90.36 eutG+] RB03 [fadBA+ betA+]0.70 4.06 0.180 0.000 0.000 90.27 RB03 ΔfadD [fadBA+ 0.72 3.98 0.2100.100 0.020 91.16 betA+] Table 2C. Synthesis of long-chain (C > 10)saturated fatty acids by RB03 derivatives Concentration (g/L) GlucoseStrain Cells utilized C10:0 C12:0 C14:0 C16:0 C18:0 % C-recovery^(b)RB03 ΔfadD 1.02 6.13 0.000 0.000 0.000 0.000 0.000 97.28 [fadBA+] RB03ΔfadD 0.72 3.07 0.000 0.000 0.000 0.700 0.180 98.14 [fadBA+ fadM+] RB03ΔfadD 0.53 2.87 0.050 0.000 0.080 0.450 0.100 91.94 [fadBA+ yciA+]^(a)Data represent averages from three samples taken from shake flaskcultures grown on 2% (w/v) glucose minimal medium for: A. 24 h, B. 48 h,and C. 72 h. ^(b)Carbon recovery was calculated by multiplying the“moles of product per mole of glucose” times the number of carbon atomsin the molecule.

TABLE 3 Strains used in this study Strains Description/Genotype SourceMG1655 F- λ- ilvG- rfb-50 rph-1 Cronon fadR atoC(c) MG1655 fadRatoC(con) Dellomonoco RB01 MG1655 fadR atoC(con) ΔarcA Δcrp::crp* Thisstudy RB02 MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdA Thisstudy RB02 ΔyqhD MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdAΔyqhD This study RB02 ΔeutE MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhEΔpta ΔfrdA ΔeutE ΔyqhD This study ΔyqhD RB02 ΔyqeF MG1655 fadR atoC(con)ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdA ΔyqeF This study RB02 ΔfadB MG1655fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdA ΔfadB This study RB02ΔydiO MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdA ΔydiO Thisstudy RB02 ΔfucO MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdAΔfucO This study RB03 MG1655 fadR atoC(con) ΔarcA Δcrp::crp* ΔadhE ΔptaΔfrdA ΔfucO ΔyqhD This study RB03 ΔfadD MG1655 fadR atoC(con) ΔarcAΔcrp::crp* ΔadhE Δpta ΔfrdA ΔfucO ΔyqhD ΔfadD This study

TABLE 4 Plasmids used in this study Plasmid Description/Genotype SourcepTrcHis2A pTrcHis2A (pBR322-derived), oriR pMB1, lacI^(q), blaInvitrogen (Carlsbad, CA) pTH fadBA E. coli fadBA genes under trcpromoter and lacI^(q) This study control in pTrcHis2A pTH yqeF E. coliyqeF gene under trc promoter and lacI^(q) control This study inpTrcHis2A pZS blank oriR pSC101*, tetR, cat, contains P_(LtetO-1)Yazdani pZS atoB E. coli atoB gene under control of P_(LtetO-1)(tetR,oriR This study SC101*, cat) pZS betA E. coli betA gene under control ofP_(LtetO-1)(tetR, oriR This study SC101*, cat) pZS eutG E. coli eutGgene under control of P_(LtetO-1)(tetR, oriR This study SC101*, cat) pZSfadB E. coli fadB gene under control of P_(LtetO-1)(tetR, oriR Thisstudy SC101*, cat) pZS fadM E. coli fadM gene under control ofP_(LtetO-1)(tetR, oriR This study SC101*, cat) pZS fucO E. coli fucOgene under control of P_(LtetO-1)(tetR, oriR This study SC101*, cat) pZSyciA E. coli yciA gene under control of P_(LtetO-1)(tetR, oriR Thisstudy SC101*, cat) pZS ydiO E. coli ydiO gene under control ofP_(LtetO-1)(tetR, oriR This study SC101*, cat) pZS yiaY E. coli yiaYgene under control of P_(LtetO-1)(tetR, oriR This study SC101*, cat) pZSyqhD E. coli yqhD gene under control of P_(LtetO-1)(tetR, oriR Thisstudy SC101*, cat)

TABLE 5 Comparison of crp, fadR, and atoSC loci of engineered strainRB02 (fadR atoC(c) ΔarcA Δcrp::crp* ΔadhE Δpta ΔfrdA) and wild-typeMG1655. Gene Accession # for Mutations/insertions locus RB02 sequence inRB02 sequence Comments crp BankIt1445305 I113L, T128I, A145T Mutationscollectively reduce dependence on cAMP for activation of Seq1 JF781281catabolic genes by CRP, as previously described^(12, 45). fadRBankIt1446148 IS5 insertion between bp Inactivation of fadR by IS5insertion, which would preclude synthesis of Seq1 JF793627 395-396 offadR gene C-terminal half of FadR and hence DNA binding. Characteristicphenotype of fadR inactivation previously confirmed in strain fadRatoC(c)¹⁰. atoSC BankIt1445920 I129S atoC tranduced from LS5218(constitutive atoC expression⁴⁶). Seq1 JF793626 Characteristic phenotypeof constitutive expression of ato operon confirmed in strain fadRatoC(c)¹⁰.

TABLE 6 Cell growth, glucose utilization, product synthesis, and carbonrecovery for wild- type and engineered strains grown on glucose minimalmedium^(a) Table 6A. Synthesis of n-butanol in wild-type and engineeredE. coli strains Concentration^(c) (g/L) Strain^(b) Cells Glucoseutilized Butanol % C-recovery^(d) MG1655 0.85 7.33 ND 88.77 fadR atoC(c)0.71 5.16 ND 85.78 RB01 (fadR atoC(c) ΔarcA crp*) 0.53 6.43 ND 90.23RB02 (RB01 ΔadhE ΔfrdA Δpta) 0.29 1.76 ND 94.09 RB02 [yqhD+] 0.49 4.270.11 91.70 RB02 [fucO+] 0.41 3.66 0.23 98.48 RB02 [yqeF+] 0.68 4.00 0.3597.13 RB02 [yqeF+ fucO+] 0.72 10.19 1.85 86.05 MG1655 [yqeF+ fucO+] 0.947.95 ND 91.15 RB02 ΔyqhD [yqeF+ fucO+] 0.49 7.89 1.90 84.15 RB02 ΔyqhDΔeutE [yqeF+ fucO+] 0.66 7.66 2.15 93.54 Table 6B. Synthesis of 4-Ccarboxylic acids by derivatives of strain RB02 Concentration^(c) (g/L)β- trans-2- hydroxy- butenoic Strain^(b) Cells Glucose utilizedβ-keto-C4:0 C4:0 acid % C-recovery^(d) RB02 0.29 1.76 ND ND ND 94.09RB02 0.62 4.25 ND 0.032 0.010 95.30 [tesA+] RB02 0.57 4.30 0.024 ND ND89.88 [tesB+] RB02 0.56 4.95 ND 0.045 0.012 91.08 [yqeF+ tesA+] RB020.42 3.34 ND 0.140 0.171 89.23 ΔydiO [yqeF+ tesA+] RB03 0.65 5.16 0.110ND ND 97.17 [yqeF+ tesB+] RB03 0.62 5.03 0.450 ND ND 95.53 ΔfadB [yqeF+tesB+] Table 6C. Synthesis of long-chain (C > 10) saturated fatty acidsby RB03 (RB02 ΔyqhD ΔfucO ΔfadD) derivatives Concentration^(c) (g/L)Extracellular FFAs (Free Fatty Acids) Glucose Total TotalFFA % C-Strain^(b) Cells utilized C10:0 C12:0 C14:0 C16:0 C18:0 FFAs^(e) s/CDWrecovery^(d) MG1655 1.29 12.88 ND ND ND ND ND ND / 93.05 RB03 0.63 4.30ND ND ND ND ND ND / 90.64 RB03 [fadBA+] 0.51 2.24 ND ND ND ND ND 0.0900.175 91.55 RB03 [fadBA+ tesA+] 0.56 5.82 ND ND 0.110 ND ND —^(f) —92.02 RB03 [fadBA+ tesB+] 0.62 5.16 0.120 ND ND ND ND —^(f) — 94.78 RB03[fadBA+ yciA+] 0.53 2.87 0.050 ND 0.080 0.450 0.100 —^(f) — 91.94 RB03[fadBA+ 0.72 3.07 ND ND ND 0.700 0.180 —^(f) — 98.14 fadM+] RB03[fadM+]^(g) 0.55 3.30 ND ND 0.020 0.150 0.080 0.435 0.790 92.86 RB03[fadBA.fadM+] 0.67 5.91 ND ND ND 0.740 0.500 1.370 2.035 97.19 MG1655ΔadhE Δpta 0.38 5.04 ND ND ND ND ND ND / 86.44 ΔfrdA ΔfadD MG1655 ΔadhEΔpta 0.76 5.07 ND ND ND ND ND 0.070 0.092 94.15 ΔfrdA ΔfadD [fadM+]MG1655 ΔadhE Δpta 0.94 5.23 ND ND ND ND ND 0.261 0.279 96.17 ΔfrdA ΔfadD[fadM+]^(g) Table 6D. Synthesis of higher chain (C > 4) n-alcohols byderivatives of strain RB03 (RB02 ΔyqhD ΔfucO ΔfadD) Concentration^(c)(g/L) Glucose Strain^(b) Cells utilized n-C6—OH n-C8—OH n-C10—OH %C-recovery^(a) MG1655 ΔadhE Δpta ΔfrdA ΔfadD [betA+] 0.65 4.16 ND ND ND91.54 RB03 [fadBA+] 0.71 5.27 ND ND ND 90.38 RB03 [fadBA+ yiaY+] 0.735.06 0.170 0.080 0.170 91.97 RB03 [fadBA+ eutG+] 0.52 4.00 0.170 0.0700.010 90.36 RB03 [fadBA+ betA+] 0.72 3.98 0.210 0.100 0.020 91.16^(a)Data represent averages for three samples taken from shake flaskcultures grown on 2% (w/v) glucose minimal medium. A. cultures weregrown at 30° C. for 24 hours; B., C., D. cultures were grown at 37° C.for 48 hours. ^(b)All genotypes are shown in Table 4. ^(c)ND, notdetecTable. Minimum detection levels are: butanol, 5.84 mg l⁻¹;β-keto-C4:0, 4.09 mg l⁻¹; β-hydroxy-C4:0, 3.03 mg l⁻¹ trans-2-butenoicacid, 9.40 mg l⁻¹; C10:0, 21.76 mg l⁻¹; C12:0, 20.45 mg l⁻¹; C14:0,27.12 mg l⁻¹; C16:0, 20.17 mg l⁻¹; C18:0, 16.42 mg l⁻¹; n-C6—OH, 24.21mg l⁻¹; n-C8—OH, 26.41 mg l⁻¹; n-C10—OH, 21.23 mg l⁻¹. ^(d)Carbonrecovery was calculated as the ratio of total moles of carbon inproducts per moles of carbon in total glucose consumed and expressed onpercentage basis. ^(e)FFAs, Free Fatty Acids ^(f)Values not measured^(g)fadM was overexpressed from medium-copy vector pTrcHis2A(Invitrogen, Carlsbad, CA).

TABLE 7 In silico identification of E. coli surrogates for higher-chain(C ≧ 4) aldehyde-forming acyl-CoA reductases and aldehyde/alcoholdehydrogenases (reaction in FIG. 1). Genes shown in bold were tested inthis study. Source of gene sequences Surrogates identified Surrogatesidentified used to identify E. coli surrogates via I-TASSER⁴⁷ viaprotein BLAST⁴⁸ Organism Accession # Gene EC # FUNCTION EC #TM-Score^(a) EC-Score^(b) Gene EC # Gene E-value^(c) Identity SimilarityIdentification of E. coli surrogates for higher-chain (C ≧ 4) fattyaldehyde-forming acyl-CoA reductase Clostridium P13604 adh1 1.1.1.—NADH-dependent 1.1.1.77 0.9404 2.2424 fucO 1.1.1.1. adhE 2.0E−91 42% 62%saccharobutylicum ⁴⁹ butyraldehyde/butanol dehydrogenase 1.1.1.1. 0.86791.8069 yiaY 1.1.1.77 fucO 5.0E−66 35% 58% adhE 1.1.1.— yiaY 2.0E−65 37%56% adhP 1.1.—.— eutG 4.0E−61 35% 54% frmA 1.1.1.— yqhD 6.0E−28 25% 46%1.1.1.6 0.7979 1.4449 gldA Pseudomonas sp. strain CF600⁵⁰ Q52060 dmpF1.2.1.10 Acetaldehyde 1.2.1.12 0.7439 1.3969 gapA 1.2.1.10 mhpF 6.0E−14679% 92% dehydrogenase Acinetobacter sp. Q8RR58 acrM 1.2.1.50 Acylcoenzyme A reductase <1.1^(b) 1.—.—.— ucpA 5.0E−20 31% 49% Strain M-1⁵¹1.—.—.— ybbO 4.0E−19 30% 50% 1.1.1.— ydfG 6.0E−18 27% 47% 1.1.1.69 idnO9.0E−18 29% 49% 1.1.1.100 fabG 9.0E−17 31% 51% Acinetobactercalcoaceticus ⁵² P94129 acrI 1.2.1.n2 Fatty acyl-CoA reductase <1.1^(b)1.1.1.100 fabG 2.0E−18 31% 53% 1.1.1.69 idnO 3.0E−17 29% 48% 1.1.1.—ydfG 1.0E−16 27% 47% 1.—.—.— ybbO 1.0E−16 28% 44% 1.—.—.— ucpA 2.0E−1631% 52% Identification of E. coli surrogates for higher-chain (C ≧ 4)aldehyde/alcohol dehydrogenases Geobacillus A4IP64 GTNG_1754 1.1.1.—Alcohol Dehydrogenase 1.1.1.202 0.9565 2.5069 yqhD 1.1.—.— eutG 3.0E−5739% 57% thermodenitrificans ⁵¹ 1.1.1.77 0.9312 2.1532 fucO 1.1.1.1 yiaY3.0E−54 33% 52% 1.1.1.1. 0.8512 1.9177 yiaY 1.1.1.77 fucO 2.0E−52 34%53% adhE 1.1.1.1. adhE 3.0E−43 34% 52% adhP 1.1.1.1 yqhD 1.0E−18 28% 46%frmA 1.1.1.6 0.7715 1.5259 gldA Pseudomonas Q00593 alkJ 1.1.99.— AlcoholDehydrogenase 1.1.99.1 betA 2.0E−104 40% 59% oleovorans ⁵⁴ Thermococcussp. ESI⁵⁵ C1IWT4 adh 1.1.1.1. Iron alcohol 1.1.1.202 0.9301 2.2696 yqhD1.1.1.1 yiaY 7.0E−40 33% 50% dehydrogenase 1.1.1.77 0.9041 1.9642 fucO1.1.1.1 yqhD 4.0E−30 30% 46% 1.1.1.1. 0.8352 1.8286 yiaY 1.1.—.— eutG3.0E−27 30% 46% adhE 1.1.1.1. adhE 9.0E−27 29% 48% adhP frmA 1.1.1.60.7800 1.3793 gldA Thermococcus hydrothermalis ⁵⁶ Y14015 1.1.1.— AlcoholDehydrogenase 1.1.1.1 yiaY 4.0E−37 31% 48% 1.1.—.— eutG 2.0E−34 31% 47%1.1.1.1. adhE 1.0E−30 30% 50% 1.1.1.77 fucO 4.0E−30 30% 45% 1.1.1.1 yqhD5.0E−19 30% 44% Sulfolobus tokodaii ⁵⁷ Q976Y8 ST0053 Hypotheticalalcohol 1.1.1.1. 0.9655 1.8286 yiaY 1.1.1.1. adhP 4.0E−33 31% 50%dehydrogenase adhE 1.—.—.— ydjJ 8.0E−26 30% 50% adhP 1.—.—.— yphC2.0E−21 31% 47% frmA 1.—.—.— yahK 2.0E−21 29% 45% 1.1.1.— rspB 6.0E−2129% 49% 1.1.1.103 tdH 9.0E−20 27% 47% 1.—.—.— yjjN 3.0E−19 26% 45%1.1.—.— gatD 1.0E−18 28% 44% ^(a)The Template Modeling-score (TM-score)is defined to assess the topological similarity of protein structurepairs. Its value ranges between 0 and 1, and a higher score indicatesbetter structural match. Statistically, a TM-score <0.17 means arandomly selected protein pair with the gapless alignment taken fromPDB⁴⁷. ^(b)An EC-score >1.1 is a good indicator of the functionalsimilarity between the query and the identified enzyme analogs⁴⁷.^(c)The BLAST E-value measures the statistical significance thresholdfor reporting protein sequence matches against the organism genomedatabase; the default threshold value is 1E−5, in which 1E−5 matcheswould be expected to occur by chance, according to the stochastic modelof Karlin and Altschul ncbi.nlm.nih.gov/BLAST/tutorial/).

Engineered Titer (g/L)/ Fermentation Carbon Medium/ Product Host Yield(% w/w)^(a) Time (hr) Source Cultivation Reference Table 8A Summary oforganisms that have been engineered to produce higher-chain (C ≧ 4)linear n-alcohols and long-chain (C ≧ 10) fatty acids Higher-chain (C ≧4) linear n-alcohols n-butanol (C4) E. coli  0.55/2.8 24 GlycerolRich/Batch 35 E. coli  0.82/3.3 100  Glucose Rich/Batch  7.8 E. coli 1.2/6.1 60 Glucose Rich-MM/HCD^(b) 58 E. coli  0.58/11.6 48 GlycerolRich/Batch 59 S. cerevisiae 0.002/0.0001 72 Galactose Rich/Batch 60 B.subtilis 0.024/0.48 72 Glycerol Rich/Batch 59 P. putida 0.120/2.4 72Glycerol Rich/Batch 59 L. lactis 0.028/— — Glucose Rich/Batch 61 L.buchneri 0.066/— — Glucose Rich/Batch 61 L. brevis  0.3/1.5 60 GlucoseRich/Batch 41 E. coli  2.05/13.4 96 Palmitic Acid MM/Fed-Batch 10 E.coli  4.65/28 72 Glucose Rich/Batch  9 E. coli  14.5/32.8 36 GlucoseMM/Batch This work n-hexanol (C6) E. coli  0.04/0.2 40 GlucoseRich/Batch 29 Fatty alcohols E. coli  0.06/0.3 — Glucose MM/Batch  2(Distribution of C10, C12, C14, and C16) Higher-chain n- E. coli 0.42/8.3 48 Glucose MM/Batch This work alcohols (C6 to C10) Table 8BSummary of organisms that have been engineered to produce higher-chain(C ≧ 4) linear n-alcohols and long-chain (C ≧ 10) fatty acids Long-chain(C ≧ 10) fatty acids Fatty Acids E. coli  1.2/6 — Glucose MM/Batch 2^(c) (Predominantly C14) Fatty Acids E. coli  2.5/— 22 GlycerolMM/Fed- 62^(c) (Wide Distribution) Batch/HCD Fatty Acids E. coli 0.81/16 29 Glycerol Rich Batch  4^(c) (Predominantly C12) Fatty AcidsE. coli  6.6/28 60 Glucose MM/Batch This work (Predominantly C16, C18)^(a)For products with carbon length distributions, titer represents thesum of products of all chain length produced. Yield assumes all thecarbon source was consumed when carbon source consumption data not givenin reference. ^(b)Two-phase, high cell density (HCD) culture grown firstin rich medium and then incubated in minimal medium (MM). ^(c)Titersreported refer to total (i.e. sum of intracellular and extracellular)free fatty acids.

TABLE 9 Homology analysis and functional annotation of E. coli ydi genesFunctional homologues Current annotation^(a) Sequence-based homologuesidentified via I-TASSER⁴⁷ Gene identified via protein BLAST⁴⁶ TM- EC-name Function Gene Function E-value^(a) Coverage Similarity FunctionScore^(c) Score^(a) ydiO Predicted acyl-CoA caiA Crotonobetainyl-CoA1.0E−102 99% 65% Butyryl-CoA 0.9584 2.2964 dehydrogenase reductasedehydrogenase aidB Isovaleryl-CoA 2.0E−14 64% 47% Acyl-CoA 0.9618 2.1730dehydrogenase dehydrogenase fadE Acyl-CoA dehydrogenase 0.001 81% 37%ydiQ Putative electron transfer fixA probable flavoprotein subunit3.0E−68 99% 71% Adenosine kinase <1.1 flavoprotein subunit required foranaerobic carnitine metabolism ydiR Putative electron transfer fixBprobable flavoprotein subunit 6.0E−75 100% 64% flavoprotein subunitrequired for anaerobic carnitine metabolism ydiS Predictedoxidoreductase fixC flavoprotein (electron 5.0E−152 100% 78%Electron-transferring- 0.9404 1.8378 with FAD/NAD(P)- transport)flavoprotein binding domain dehydrogenase ygcN predicted oxidoreductasewith 9.0E−93 99% 62% FAD/NAD(P)-binding domain ydiT Ferredoxin-likeprotein fixX putative ferredoxin possibly 3.0E−33 96% 62%Electron-transferring- 0.9006 1.5362 involved in anaerobic flavoproteincarnitine metabolism dehydrogenase ygcO predicted 4Fe—4S cluster-1.0E−18 89% 62% containing protein ^(a)As annotated in Ecocyc⁶³. Alsoreported by Campbell, J. W. and coworkers⁶⁴. ^(b)The BLAST E-valuemeasures the statistical significance threshold for reporting proteinsequence matches against the organism genome database; the defaultthreshold value is 1E−5, in which 1E−5 matches would be expected tooccur by chance, according to the stochastic model of Karlin andAltschul ncbi.nlm.nih.gov/BLAST/tutorial/). ^(c)The TemplateModeling-score (TM-score) is defined to assess the topologicalsimilarity of protein structure pairs. Its value ranges between 0 and 1,and a higher score indicates better structural match. Statistically, aTM-score <0.17 means a randomly selected protein pair with the gaplessalignment taken from PDB⁴⁷. ^(d)An EC-score >1.1 is a good indicator ofthe functional similarity between the query and the identified enzymeanalogs⁴⁷.

TABLE 10 Thermodynamic analysis of the engineered reversal of theβ-oxidation cycle Standard ΔG_(r) (Min ΔG_(r), Max ΔG_(r)) Reactionnumber and enzyme name (gene) [kcal/mol] ^(a) {circle around (1)}Thiolase (yqeF, fadA) 7.1 2 Acetyl-CoA → Acetoacetyl-CoA + CoA-SH (−1.9,16.1) {circle around (2)} Hydroxyacyl-CoA dehydrogenase (fadB) −3.7 Acetoacetyl-CoA + NADH + H⁺ → (−12.7, 5.3)  3-hydroxybutyryl-CoA + NAD⁺{circle around (3)} Enoyl-CoA hydratase (fadB) 2.1 3-hydroxybutyryl-CoA→ Crotonyl-CoA + H₂O (−2.4, 6.6)  {circle around (4)} Acyl-CoAdehydrogenase (coupled to 5.7 ubiquinone, fadE)^(b) (−3.3, 14.7)Crotonyl-CoA + UQH₂ → Butyryl-CoA + UQ {circle around (4)} Enoyl-CoAreductase (coupled to ferredoxin, −16.5  ydiO-ydiQRST)^(b) (−25.5,−7.5)  Crotonyl-CoA + Fd² → Butyryl-CoA + Fd Operation of β-oxidationreversal coupled to 11.2  ubiquinone Operation of β-oxidation reversalcoupled to −11.0  ferredoxin ^(a) Standard ΔG of formation valuesestimated using the group contribution method⁶⁵ and used to calculatethe standard ΔG of reaction⁶⁶. Minimum and maximum ΔG_(r) valuescalculated assuming standard conditions (298.15 K, pH 7) with minimumand maximum metabolite concentrations set to 0.00001M and 0.02M,respectively⁶⁶. Listed ΔG_(r) values are in good agreement withexperimentally measured/calculated ΔG_(r) values^(67,) ⁶⁸.^(b)Calculation of ΔG_(r) for enoyl-CoA reductase used standardreduction potentials from Thauer et al⁶⁷.

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What is claimed is:
 1. An engineered microorganism, wherein the microorganism is engineered to overexpress β-oxidation cycle enzymes and one or more termination enzymes as compared to a corresponding wild-type microorganism, wherein said β-oxidation cycle enzymes comprise: a) a thiolase catalyzing the conversion of (C_(n))-acyl CoA to β-ketoacyl-CoA, b) a hydroxyacyl-CoA dehydrogenase catalyzing the conversion of β-ketoacyl-CoA to β-hydroxyacyl-CoA, c) an enoyl-CoA hydratase catalyzing the conversion of β-hydroxyacyl-CoA to trans-Δ2-Enoyl-CoA, and d) an acyl-CoA dehydrogenase or transenoyl-CoA reductase catalyzing the conversion of trans-Δ2-enoyl-CoA to (C_(n+2))-acyl CoA, wherein said β-oxidation cycle runs in the reverse/biosynthetic direction, as recited in enzyme reactions a) through d), and wherein said one or more termination enzymes are selected from the group consisting of an alcohol-forming coenzyme-A thioester reductase; an aldehyde-forming CoA thioester reductase and an alcohol dehydrogenase; a thioesterase; an acyl-CoA:acetyl-CoA transferase; a phosphotransacylase and a carboxylate kinase; an aldehyde-forming CoA thioester reductase and an aldehyde decarbonylase; and OleA, OleB, OleC, and OleD.
 2. An engineered microorganism comprising a genotype, wherein said genotype comprises fadR, atoC(c), ΔarcA, Δcrp, crp*.
 3. The engineered microorganism of claim 2, wherein said genotype is selected from the group consisting of: a) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, [tesB+]; b) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, [tesB+, yqeF+]; and c) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, ΔfadB, [tesB+, yqeF+].
 4. The engineered microorganism of claim 2, wherein said genotype is selected from the group consisting of: a) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, [tesA+]; b) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, [tesA+, yqeF+]; and c) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔldhA, ΔmgsA, ΔydiO, [tesB+, yqeF+].
 5. The engineered microorganism of claim 2, wherein said genotype is selected from the group consisting of: a) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, ΔydiO, [yqeF+, tesB+]; b) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [fadBA+, fadM+]; and c) fadR, atoC(c), ΔarcA, Δcrp, crp, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [fadBA+, yciA+].
 6. The engineered microorganism of claim 2, wherein said genotype is selected from the group consisting of: a) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [yqeF+, acrM+, PCC7942_orf1593+]; b) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [yqeF+, acrl+, PCC7942_orf1593+]; c) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [yqeF+, oleABCD+]; d) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [fadBA+, acrM+, PCC7942_orf1593+]; e) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [fadBA+, acrl+, PCC7942_orf1593+]; and f) fadR, atoC(c), ΔarcA, Δcrp, crp*, ΔadhE, ΔfrdA, Δpta, ΔyqhA, ΔfucO, ΔfadD, [fadBA+, oleABCD+].
 7. A method of making a product, said method comprising growing the engineered microorganism of claim 1 under microaerobic (<10% O₂) or anaerobic conditions in a glucose minimal medium supplemented with 0-100 μM FeSO₄ and 0-5 mM calcium pantothenate at a temperature of 30-40° C. for a time sufficient to allow production of a product and isolating said product, wherein said product is selected from the group consisting of trans Δ2 fatty alcohols, β-keto alcohols, 1-3 diols, β-hydroxy acids, carboxylic acids, and hydrocarbons. 